The 1.8 Å resolution structure of hydroxycinnamoyl‐coenzyme A hydratase‐lyase (HCHL) from Pseudomonas fluorescens , an enzyme that catalyses the transformation of feruloyl‐coenzyme A to vanillin

The crystal structure of hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL) from Pseudomonas fluorescens AN103 has been solved to 1.8 Å resolution. HCHL is a member of the crotonase superfamily and catalyses the hydration of the acyl‐CoA thioester of ferulic acid [3‐(4‐hydroxy‐3‐methoxy‐phenyl)prop‐2‐enoic acid] and the subsequent retro‐aldol cleavage of the hydrated intermediate to yield vanillin (4‐hydroxy‐3‐methoxy‐benzaldehyde). The structure contains 12 molecules in the asymmetric unit, in which HCHL assumes a hexameric structure of two stacked trimers. The substrate, feruloyl‐CoA, was modelled into the active site based on the structure of enoyl‐CoA hydratase bound to the feruloyl‐CoA‐like substrate 4‐( N , N ‐dimethylamino)‐cinnamoyl‐CoA (PDB code 1ey3 ). Feruloyl‐CoA was bound in this model between helix 3 of the A subunit and helix 9 of the B subunit. A highly ordered structural water in the HCHL structure coincided with the thioester carbonyl of feruloyl‐CoA in the model, suggesting that the oxyanion hole for stabilization of a thioester‐derived enolate, characteristic of coenzyme‐A dependent members of the crotonase superfamily, is conserved. The model also suggested that a strong hydrogen bond between the phenolic hydroxyl groups of feruloyl‐CoA and B Tyr239 may be an important determinant of the enzyme's ability to discriminate between the natural substrate and cinnamoyl‐CoA, which is not a substrate.